Transient receptor potential (TRP) channels have emerged as cellular sensors of various internal and external cues. The canonical TRP (TRPC) channels are involved in receptor and store-operated Ca2+ entry, an important component of phospholipase C-activated Ca2+ signaling cascade. Under resting potentials, hese channels also conduct Na+ influx, allowing cells to be depolarized in a receptor-operated manner. TRPC channels have been implicated in the regulation of many functions, including smooth muscle and vascular tones in blood vessels, endothelial permeability, fertilization, saliva secretion, synaptictransmission, and neurite outgrowth. Our long-term objective is to understand the mechanism of regulation of Ca2+ signal nside cells and its roles in human health. However, the lack of specific agonists and antagonists has seriously hindered the studies on the characterization of physiological functions as well as the mechanisms of regulation of different TRP channels. The biological research community has an urgent need for pharmacological tools that are specifically targeted at different TRP members. Moreover, several TRP ;hannels have been implicated in human diseases. The development of specific drugs for TRP channels could also have therapeutic implications. The goals of the current proposal are to develop microplate-based assays to monitor TRP channel function and to configure them for high throughput screening. Emphasis will be placed on TRPC channels with the use of fluorescence-based membrane potential assays. A selected number of TRPV and TRPM channels will also be used for counterscreens. Intracellular Ca2+ measurement will be used as secondary screens and whole cell voltage clamp experiments will be used for final verification of the active compounds. Two specific aims are proposed. AIM I will explore conditions that give rise to the best signal-to-noise ratio, high consistency, and optimal reproducibility of receptor-induced activation of TRPC channels stably expressed in HEK293 cells in microplate-based fluorescence membrane potential assays. AIM II will configure the assays for high throughput screening. Pilot screens on TRPC5 and TRPC6 will be performed using analogs of 2-aminoethoxydiphenyl borate and classical L-type Ca2+ channel blockers. The configured assay conditions and cell lines are to be used in the Molecular Libraries Screening Centers Network to screen for ligands of TRPC channels.